Vol-3/Issue-1/Jan-Feb 2014 PhTechMed ISSN: 2278-1099

Review on Analgesic Activity and Determination Methods

Amol S. Deshmukh*, Pravin G. Morankar and Manoj R. Kumbhare.
SMBT College of Pharmacy, Nandi Hills, Dhamangaon, Nasik, India

Abstract
Pain is a designation for a spectrum of sensations of highly divergent character and intensity ranging from unpleasant to intolerable.
The struggle to relieve pain began with the origin of humanality. An analgesic may be defined as a drug bringing about insensibility to
pain without loss of consciousness. Analgesic drugs act in various ways on the peripheral and central nervous systems. They are
distinct from anesthetics, which reversibly eliminate sensation, and include Paracetamol [known in the US as Acetaminophen or
simply APAP], the non-steroidal anti-inflammatory drugs [NSAIDs] such as the salicylates, and opioid drugs such as morphine and
opium. In choosing analgesics, the severity and response to other medication determines the choice of agent; the World Health
Organization [WHO] pain ladder. By reviewing methods for analgesic drugs we found that mainly chemical and physical methods are
available. As pain is the first indication of most of diseases like neuropathic pain, chest pain, pain in stomach and various organs
related pain are involved. Only six methods are mostly used for determination of analgesic activity there is scope to find alternative
methods.

Key Words: Analgesic, Opioid, Morphine, Paracetamol

Introduction
An analgesic, or painkiller, is any member of the group of
drugs used to achieve analgesia-relief from pain [Tambaro,
2013]. Analgesic drugs act in various ways on the peripheral
and central nervous systems. They are distinct from
anesthetics, which reversibly eliminate sensation, and include
Paracetamol [known in the US as Acetaminophen or simply
APAP], the non-steroidal anti-inflammatory drugs [NSAIDs]
such as the salicylates, and opioid drugs such as morphine and
opium. In choosing analgesics, the severity and response to
other medication determines the choice of agent; the World
Health Organization [WHO] pain ladder [Harper, 2001]
specifies mild analgesics as its first step. Analgesia/Pain is ill-
defined unpleasant sensation evoked by stimulus [external /
internal] - the most important symptom giving warning signal
and primarily protective in nature. Analgesia due to blockade
of pain nerve sensitizing mechanism induced by bradykinin,
TNFα, ILs [Deng, 2011]. An analgesic is a drug that
selectively relieves pain by acting in the CNS or on peripheral
pain mechanisms, without significantly altering consciousness.
Pain is a warning signal, primarily protective in nature, but
causes discomfort and suffering; may even be unbearable and
incapacitating. Excessive pain may produce other effects-
sinking sensation, apprehension, sweating, nausea, palpitation,
rise or fall in BP, tachypnoea. Analgesics relieve pain as a
symptom, without affecting its cause [Tripathi, 2008].
Pain Mechanisms and Pathways
Pain is a designation for a spectrum of sensations of highly
divergent character and intensity ranging from unpleasant to
intolerable. Pain stimuli are detected by physiological
receptors [sensors, nociceptors] least differentiated
morphologically, viz., free nerve endings. The body of the
bipolar afferent first-order neuron lies in a dorsal root
ganglion. Nociceptive impulses are conducted via
unmyelinated [C-fibers, conduction velocity 0.2–2.0 m/s] and
myelinated axons [Aδ-fibers, 5–30 m/s]. The free endings of
Aδ fibers respond to intense pressure or heat, those of C-fibers
respond to chemical stimuli [H+, K+, histamine, bradykinin,
etc.] arising from tissue trauma.
Address for Correspondence
Email ID: meamoldeshmukh@rediffmail.com
Tel No.: +91-9371393020
Received: 20/12/2013
Accepted: 10/02/2014
www.pharmtechmedica.com
Irrespective of whether chemical, mechanical, or thermal
stimuli are involved, they become significantly more effective
in the presence of prostaglandins. Chemical stimuli also
underlie pain secondary to inflammation or ischemia [angina
pectoris, myocardial infarction],or the intense pain that occurs
during over distention or spasmodic contraction of smooth
muscle abdominal organs and that may be maintained by local
anoxemia developing in the area of spasm [visceral pain]. Aδ
and C-fibers enter the spinal cord via the dorsal root, ascend in
the dorsolateral funiculus, and then synapse on second-order
neurons in the dorsal horn. The axons of the second-order
neurons cross the midline and ascend to the brain as the
antero-lateral pathway or spinothalamic tract. Based on
phylogenetic age, neo- and paleospinothalamic tracts are
distinguished. Thalamic nuclei receiving neospinothalamic
input project to circumscribed areas of the postcentral gyrus.
Stimuli conveyed via this path are experienced as sharp,
clearly localizable pain. The nuclear regions receiving
paleospinothalamic input project to the postcentral gyrus as
well as the frontal, limbic cortex and most likely represent the
pathway subserving pain of a dull, aching, or burning
character, i.e., pain that can be localized only poorly. Impulse
traffic in the neo- and paleospinothalamic pathways is subject
to modulation by descending projections that originate from
the reticular formation and terminate at second-order neurons,
at their synapses with first-order neurons, or at spinal
segmental inter neurons [descending antinociceptive system].
This system can inhibit impulse transmission from first- to
second-order neurons via release of opiopeptides [enkephalins]
or monoamines [norepinephrine, serotonin] [Heinz, 1996].
Pain sensation can be influenced or modified as follows:
 Elimination of the cause of pain.
 Lowering of the sensitivity of nociceptors [antipyretic
analgesics, local anesthetics].
 Interrupting nociceptive conduction in sensory nerves
[local anesthetics].
 Suppression of transmission of nociceptive impulses in
the spinal medulla [opioids].
 Inhibition of pain perception [opioids, general
anesthetics].
 Altering emotional responses to pain, i.e., pain behavior
[Heinz, 1996].
425
Vol-3/Issue-1/Jan-Feb 2014
Opioid Analgesics and Antagonists
Morphine, the prototypical opioid agonist, has long been
known to relieve severe pain with remarkable efficacy. The
opium poppy is the source of crude opium from which
Serturner in 1803 isolated the pure alkaloid morphine named
after Morpheus, the Greek god of dreams. It remains the
standard against which all drugs that have strong analgesic
action are compared. These drugs are collectively known as
"opioid analgesics" and include not only the natural and
semisynthetic alkaloid derivatives from opium but also include
synthetic surrogates, other opioid-like drugs whose actions are
blocked by the nonselective antagonist naloxone, plus several
endogenous peptides that interact with the several subtypes of
opioid receptors.
Clinical Uses of Opioid Analgesics
1. Analgesia
Severe, constant pain is usually relieved with opioid
analgesics with high intrinsic activity whereas sharp,
intermittent pain does not appear to be as effectively
controlled. The pain associated with cancer and other
terminal illnesses must be treated aggressively and often
requires a multidisciplinary approach for effective
management. Such conditions may require continuous use of
potent opioid analgesics and will be associated with some
degree of tolerance and dependence. However, this should
not be used as a barrier to providing patients with the best
possible care and quality of life. Research in the hospice
movement has demonstrated that fixed interval
administration of opioid medication [i.e, a regular dose at a
scheduled time] is more effective in achieving pain relief
than dosing on demand. New dosage forms of opioids that
allow slower release of the drug are now available. Their
purported advantage is a longer and more stable level of
analgesia. If disturbances of gastrointestinal function prevent
the use of oral sustained-release morphine, the fentanyl
transdermal system [fentanyl patch] can be used over long
periods. Furthermore, buccal transmucosal fentanyl can be
used for episodes of breakthrough pain. Administration of
strong opioids by nasal insufflation has been shown to
beefficacious, and nasal preparations are now available in
some countries. Approval of such formulations in the USA
is growing. In addition, stimulant drugs such as the
amphetamines have been shown to enhance the analgesic
actions of the opioids and thus may be very useful adjuncts
in the patient with chronic pain. Opioid analgesics are often
used during obstetric labor. Because opioids cross the
placental barrier and reach the fetus, care must be taken to
minimize neonatal depression. If this occurs, immediate
injection of the antagonist naloxone will reverse the
depression. The phenylpiperidine drugs [e.g., meperidine]
appear to produce less depression, particularly respiratory
depression, in newborn infants than does morphine; this may
justify their use in obstetric practice. The acute, severe pain
of renal and biliary colic often requires a strong agonist
opioid for adequate relief. However, the drug-induced
increase in smooth muscle tone may cause a paradoxical
increase in pain secondary to increased spasm. An increase
in the dose of opioid is usually successful in providing
adequate analgesia.
2. Acute Pulmonary Edema
The relief produced by intravenous morphine in dyspnea
from pulmonary edema associated with left ventricular
failure is remarkable. The mechanism is not clear but
probably involves reduced perception of shortness of breath
and reduced patient anxiety as well as reduced cardiac
preload [reduced venous tone] and after load [decreased
www.pharmtechmedica.com
Deshmukh et al.,
peripheral resistance]. Morphine can be particularly useful
when treating painful myocardial ischemia with pulmonary
edema.
3. Cough
Suppression of cough can be obtained at doses lower than
those needed for analgesia. However, in recent years the use
of opioid analgesics to allay cough has diminished largely
because a number of effective synthetic compounds have
been developed that are neither analgesic nor addictive.
These agents are discussed below.
4. Diarrhea
Diarrhea from almost any cause can be controlled with the
opioid analgesics, but if diarrhea is associated with infection
such use must not substitute for appropriate chemotherapy.
Crude opium preparations [e.g., paregoric] were used in the
past to control diarrhea, but now synthetic surrogates with
more selective gastrointestinal effects and few or no CNS
effects, e.g., diphenoxylate, are used. Several preparations
are available specifically for this purpose.
5. Applications in Anesthesia
The opioids are frequently used as premedicant drugs before
anesthesia and surgery because of their sedative, anxiolytic,
and analgesic properties. The opioids are also used
intraoperatively both as adjuncts to other anesthetic agents
and, in high doses [e.g., 0.02–0.075 mg/kg of fentanyl], as a
primary component of the anesthetic regimen, most
commonly in cardiovascular surgery and other types of
high-risk surgery where a primary goal is to minimize
cardiovascular depression. In such situations, mechanical
respiratory assistance must be provided [Bertram, 2003].
Methods for Determination of Analgesic Activity
1. Acetic Acid-Induced Writhing Test
Acetic acid induced writhing model was used to evaluate
analgesic activity of the synthesized compounds. Five
groups of six Swiss albino mice, each 20–25 g b.w, were
used. 0.6% acetic acid [dose¼ 10 ml/Kg] was injected intra-
peritoneally. The numbers of writhes were counted for 20
min, after 5 min of injection of acetic acid into each mice.
This reading was taken as a control. Next day, same groups
of mice were used for evaluating analgesic activity. Each
group was administered orally with the synthesized
compounds. The dose of 100 mg/kg of animal was given 1
hour before injection of acetic acid. After 5 min of acetic
acid injection, mice were observed for the number of
writhings for the duration of 20 min. The mean value for
each group was calculated and compared with control
[Kavitha, 2010; Hoonur, 2010].
2. Hot Plate Test
The hot plate test was carried out at a fixed temperature of
55 ± 0.5◦C. Animals were habituated twice to the hot plate
in advance. Response was defined as licking or biting of a
paw, or jumping [where all four paws leave the plate]. The
time in seconds between the platform and reaction was
recorded as the response latency. The mice exhibiting
latency time greater than 30s or less than 5s were excluded.
The latency time was determined at 30 min, 60 min, 90 min,
and 120 min after administration of the test drugs,
Meperidine hydrochloride, and saline. A latency period of
60 s was defined as complete analgesia, and if so, we will
cut off its time to prevent damage to mice. After each
testing, the hot plate was wiped clean with wet paper towels,
removing urine and faeces. The female ICR mice were
divided into seven groups, control group with i.g.
isometrical physiological saline, and the mice were
administered test drugs and positive drug for three days. The
426
Vol-3/Issue-1/Jan-Feb 2014
positive control group was received dolatin injection [25
mg/kg] before placed on hot plate [Sua, 2011; Chen, 2012].
3. Tail Flick Test
The apparatus used in the tail flick test consisted of a
circulating immersion water heater. The thermostat was
adjusted so that a constant temperature of 54±1 ◦C was
maintained in the water bath. Before treatment, the terminal
3 cm of each mouse’s tail was immersed in the water bath
and the time in seconds taken to flick the tail was recorded.
Only mice showing a pre-treatment reaction time less or
equal to 3s were selected for the study. Immediately after
basal latency assessment, the plant extracts, reference
substance or solvent were administered by the oral route to
groups of 5 mice and the reaction time was again measured
1 and 2 h after the injections. Cut-off time was 6 s for tail-
flick measurements in order to minimize tissue injury
[Rabanal, 2005; Sanchez-Mateo, 2006].
4. Tail Pressure Test
In the present study, the tail pressure test was performed
according to the procedures with minor modification.
Different alkaloids dissolved in hydrogel were topically
administered to the tail of the mice at a dose of 6 mg/kg. The
threshold of the motor response to pressure applied to the
tail by an Analgesy Meter was measured. Using this device,
the distal part of the tail was supported by a plinth while
linearly increasing pressure applied with a cone-shaped
pusher. The mechanical nociceptive threshold was defined
as the force at which the mice withdrew its tail or struggled
60 min after administration. The cut-off pressure was 500 g
to avoid tissue damage [Chen, 2012].
5. Tail Formalin Test
A modified formalin test was used. The different extracts at
5% [w/v] concentration were applied by topical
administration as described in the tail flick test, and mice
were immediately intradermically injected 20uL of a
formaldehyde solution to 10% into dorsal surface of their
tail, using a tuberculin syringe. Then the mouse was located
into a chamber, with mirrors walls to enable clear
observation of animal tails for 5 min. The nociceptive
behavior is directly proportional to the licking time of the
tail, which is a monophasic process. The time-course
observation was restricted to 5 min, the length of time
during which pain occurs. Antinociceptive activity [A] was
expressed according to the following formula: A [%] =
100−[[T1×100]/T2], where T1 stands for the mean licking
time post extract and T2 is the mean licking time control.
For each concentration of the different extracts, the topic
analgesic activity was evaluated using groups of 8 animals
for a single dose and groups of 16 control animals were
similarly treated, but they did not receive the extracts.
Another one group of eight animals were pretreated with
ibuprofen, reference drug at 5, 2.5, 1.2 and 0.6% [w/v]
concentrations [Delporte, 2007].
6. Formalin Test
Control group received 5% formalin. Twenty microliter of
5% formalin was injected into the dorsal surface of the right
hind paw 60 min after administration of WET [0.1, 0.5, 1.0
and 2.0 g/kg, p.o.] and 30 min after administration of Indo
[10 mg/kg, i.p.]. The mice were observed for 30 min after
the injection of formalin, and the amount of time spent
licking the injected hind paw was recorded. The first 5 min
post formalin injection is referred to as the early phase and
the period between 15 and 40 min as the late phase. The
total time spent licking or biting the injured paw [pain
www.pharmtechmedica.com
Deshmukh et al.,
behavior] was measured with a stop watch. The activity was
recorded in 5 min intervals [Sheu, 2009; Deng, 2011;
Tambaro, 2013].
Conclusion
Analgesics and the methods for determination of analgesic
activity were shortly described. It is found that there are
mainly six methods that are mostly used for the determination
of analgesic activity.
References
Kavitha CS, Hosamani KM, Seetharamareddy HR [2010]. In-
vivo analgesic and anti- inflammatory activities of newly
synthesized Benzimidazole derivatives, European Journal
of Medicinal Chemistry 45: 2048–2054.
Hoonur RS, Patil BR, Badiger DS, Vadavi RS, Gudasi KB,
Dandawate PR, Ghaisas MM, Padhye SB, Nethaji M
[2010]. Transition metal complexes of 3-aryl-2-substituted
1,2-dihydroquinazolin-4[3H]-one derivatives: New class of
analgesicand anti-inflammatory agents, European Journal
of Medicinal Chemistry 45: 2277–2282.
Rabanal RM, Bonkanka CX, Hern M and ez-P´erez,
CCS´anchez-Mateo [2005]. Analgesic and topical anti-
inflammatory activity of HypericumcanarienseL. And
HypericumglandulosumAit. Journal of Ethnopharmacology
96: 591–596.
Sanchez-Mateo CC, Bonkanka CX, Hern M and ez-P´erez,
RM Rabanal [2006]. Evaluation of the analgesic and
topical anti-inflammatory effects of HypericumreflexumL.
fil, Journal of Ethnopharmacology 107: 1–6.
Sua S, Wang T, Duan J, Zhou W, Hua YQ, Tang YP, Qian
DW [2011]. Anti-inflammatory and analgesic activity of
different extracts of Commiphoramyrrha, Journal of
Ethnopharmacology 134: 251–258.
Chen J, Wang X, Qua Y, Chen Z, HaoCai, Xiao L, FeiXu, Tu-
lin L, Bao-changCai [2012]. Analgesic and anti-
inflammatory activity and pharmacokinetics of alkaloids
from-seeds of Strychnos nux-vomica after transdermal
administration: Effect of changes in alkaloid composition,
Journal of Ethnopharmacology 139: 181– 188.
Delporte C, Backhouse N, Inostroza V, Aguirre MC, Peredo
N, Silva X, Negrete R, Miranda HF [2007]. Analgesic
activity of Ugnimolinae [murtilla] in mice models of acute
pain, Journal of Ethnopharmacology 112: 162–165.
Sheu MJ, Chou PY, Cheng HC, Wu CH, Huang GJ, Wang BS,
Chen JS, Chien YC, Huang MH [2009]. Analgesic and
anti-inflammatory activities of a water extract of
Trachelospermum jasminoides [Apocynaceae], Journal of
Ethnopharmacology 126: 332–338.
Deng JS, Chi CS, Huang SS, Shie PH, Lin TH, Huang GJ
[2011]. Antioxidant, analgesic, and anti-inflammatory
activities of the ethanolic extracts of Taxillus liquid
ambaricola, Journal of Ethnopharmacology 137: 1161–
1171.
Saif SR [2005]. Pharmacology Review, First edition, CBS
Publishers & Distributors: pp. 177, 206.
Tripathi KD [2008]. Essentials of Medical Pharmacology,
Sixth edition, Jaypee brothers medical publishers [p] ltd.
pp.453.
427
Vol-3/Issue-1/Jan-Feb 2014 Deshmukh et al.,
Tambaro S, Reali R, Volonterio A, Zanda M, Olimpieri F,
Pinna GA, Lazzari P [2013] NESS002 ie: A new
fluorinated thiolendopeptidase inhibitor with
antinociceptive activity in an animal model of persistent
pain, Pharmacology, Biochemistry and Behavior, pp. 1-7.
Harper D [2001]. Online Etymology Dictionary: Analgesia.
Retrieved December 3:2012.
Anonymous [1990]. Cancer pain relief and palliative care;
report of a WHO expert committee. World Health
Organization Technical Report Series, 804. Geneva,
Switzerland: World Health Organization. pp. 1–75.
Bertram G. Katzung [2003]. Basic & Clinical Pharmacology,
9th Edition, pp. 691-695.
Heinz L, Ziegler A, Mohr K, Bieger D [1996]. Color Atlas of
Pharmacology, Third edition, pp.194.

www.pharmtechmedica.com 428